Complete nucleotide sequence and analysis of Planobispora linear plasmid pPR2

Abstract:

[Objective] To obtain the complete nucleotide sequence of Planobispora linear plasmid pPR2, and reveal the novel telomere replication protein and possible replication loci from the centrally located origin. [Methods] We cloned the EcoRI DNA fragments and assembled them to get the whole sequence of pPR2. We analyzed the secondary structure of the telomere DNA and the putative telomere replication protein using software, and detected the possible replication loci by Streptomyces protoplast transformation. [Results] The complete nucleotide sequence of pPR2 consists of 15520 bp in length, with a 68.1% (G+C) content. The 329 bp terminal inverted repeat sequence can’t form the conserved fold-back secondary structure as that of many Streptomyces linear replicons. Lacking of typical Streptomyces tap/tpg locus for te-lomere replication, pPR2.3c encodes a protein with two domains resembling the telomere associated protein of Strepto-myces and helicase of Haemophilus respectively. No typical Streptomyces iteron-rep locus for replication from the cen-trally located origin, two DNA fragments containing almost all pPR2 were cloned and introduced by transformation into S. lividans ZX7, but no transformants were obtained. pPR2 encodes single-strand binding protein which may regulate repli-cation of linear DNA and Tra protein for conjugation transfer in actinomycetes. [Conclusion] pPR2 is the smallest ac-tinomycete linear plasmid beyond Streptomyces. This is the first time to report the complete nucleotide sequence of linear plasmid in the genus Planobispora. pPR2 may have novel mechanism for telomere replication, and pPR2.2c and pPR2.3c encode the possible telomere replication proteins.